The solubility of this substrate in aqueous medium is not very high. One site gave it as 0.3 mg/ml in phosphate-buffered saline at pH 7.2. However, another site gave its solubility as 10 mg/ml in water. My surmise from these data points is that the solubility is better if the ionic strength of the solution is decreased. Therefore, instead of using 0.1 M phosphate, try reducing the buffer concentration to, say, 20 mM, to increase the substrate's solubility. Do not attempt to use a concentration higher than the solubility.

Make sure you are measuring at the optimal wavelength, which is probably around 405-410 nm. At this wavelength, the color generated will be pale yellow unless quite a lot of product is present.

Compounds that you wish to test may also absorb at the same wavelength as the product of the reaction, either because the compound is colored, or because it is insoluble and causing the solution to be cloudy. You can easily check for this problem by measuring the absorbance of the compound without adding the enzyme or substrate, just with the same volume of the buffer.

If you make a continuous measurement of the enzyme reaction (for example, remeasuring every minute), you will be able to measure the slope of the reaction progress curve. Since the slope is not affected by a minor background absorbance from the compound, you can use the slope to measure the enzyme activity even if the compound has a bit of color.

Thank you Mr Adam. I will try it again with reduced buffer concentration.